Bispecific antibodies targeting intracellular ny-eso-1 and uses thereof

Abstract

The application discloses a bispecific antibody targeting intracellular NY-ESO-1 and application thereof, and relates to the technical field of biological medicines. The bispecific antibody targeting intracellular NY-ESO-1 is designed, a symmetric form retains a complete molecular structure of a CD3 monoclonal antibody, and an anti-NY-ESO-1 157‑165 / A02 nanobody sequence is added at an N terminal, and the bispecific antibody can specifically recognize two different antigens. The bispecific antibody provided by the application has target point dependence, can significantly cause up-regulation of T cell early activation markers and late activation markers, and has obvious specific in-vivo anti-tumor activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bispecific antibody targeting intracellular NY-ESO-1 and its applications. Background Technology

[0002] In recent years, the incidence of cancer has been rising, seriously threatening human health. Cancer immunotherapy is a novel therapy that has developed rapidly over the past decade, progressing alongside chemotherapy, targeted therapy, surgery, and radiotherapy. Under normal circumstances, the immune system can recognize and eliminate tumor cells. However, tumor cells possess the ability to evade the immune system, inhibiting the immune system's effective recognition and killing of tumor cells through various mechanisms, thereby generating immune tolerance and even promoting tumor development and progression. Cancer immunotherapy primarily targets the body's immune system rather than tumor cells, eliminating them by enhancing the body's natural immune defense against tumors and reshaping the immune microenvironment. This therapy, on the one hand, trains immune cells to recognize and eliminate target cells carrying tumor antigens, enhancing immune-mediated tumor cell lysis; on the other hand, it eliminates or reduces immunosuppressive signals induced by tumor cells, thereby achieving the therapeutic effect of cancer treatment.

[0003] Bispecific antibodies (BiAbs) are artificial antibodies composed of two different antibody fragments that can specifically recognize and bind to two different antigens or two different antigenic epitopes. In the complex pathogenesis of cancer, multiple mediators are involved in the activation of cancer-related signaling pathways, which limits the effectiveness of single-specific cancer therapies. A reduction in activated lymphocytes in the tumor microenvironment (TME) has been shown to lead to adverse immune responses. The mechanisms of action of bispecific antibodies in cancer immunotherapy include: 1) binding to T cells or other immune cells (such as natural killer cells) to specifically eliminate tumor cells; 2) bridging receptors to block or activate synergistic signaling pathways; and 3) targeting multiple tumor antigens or different antigenic epitopes on tumor cells to improve tumor selectivity (Li H, Er Saw P, Song E. Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020, 17(5): 451-461.).

[0004] NY-ESO-1 is a type of tumor-associated testicular antigen, first discovered by Chen et al. through serological identification of esophageal squamous cell carcinoma patients. It is a tumor-shared antigen capable of inducing humoral immunity. NY-ESO-1 exhibits high expression levels in neuroblastoma, synovial sarcoma, esophageal cancer, and lung cancer, with expression increasing with disease progression. In rectal cancer and lymphoma, expression is very low or even absent, but expression is restricted in normal tissues. While theoretically classified as a tumor-associated antigen, because the testes / placenta do not express HLA alleles, the peptides produced from NY-ESO-1 cannot be presented to the cell surface. Therefore, NY-ESO-1 can be considered a tumor-specific antigen. Studies have found that NY-ESO-1 can induce both humoral and immunization, with serum IgG positivity and the production of CD8 antibodies against this antigen being associated with this antigen. + T cells are positively correlated, and it is the most immunogenic tumor-specific antigen discovered to date (Raza A, Merhi M, Inchakalody VP, et al. Unleashing the immune response to NY-ESO-1 cancer testis antigen as a potential target for cancer immunotherapy. J Transl Med, 2020, 18(1): 140.)

[0005] The recruitment of effector cells is crucial in tumor immunotherapy. One arm of a bispecific antibody targets receptors on the surface of tumor cells, while the other arm binds to CD3ε in the TCR complex, recruiting and activating T cells, further inducing highly efficient and selective cytotoxicity. When both T cells and tumor cells are simultaneously bound by a bispecific antibody, it mediates the formation of a cytolytic synapse between T cells and tumor cells. This synapse contains perforin and cytotoxic granzyme B released by the T cell, leading to tumor cell killing. The main functions of the CD3 molecule are: stabilizing the TCR structure and transmitting T cell activation signals. When the TCR specifically recognizes and binds to an antigen, CD3 participates in transducing the signal into the T cell cytoplasm, serving as the first signal to induce T cell activation and playing a vital role in T cell antigen recognition and immune response. Bispecific antibodies targeting CD3 are rapidly becoming the most revolutionary approach in cancer immunotherapy, accounting for more than 50% of bispecific antibody drugs used in global clinical research for cancer treatment. Summary of the Invention

[0006] Therefore, this invention provides a bispecific antibody targeting intracellular NY-ESO-1 and its application. This invention designs a bispecific antibody targeting intracellular NY-ESO-1, which retains the complete molecular structure of a CD3 monoclonal antibody in a symmetrical form, while adding anti-NY-ESO-1 at the N-terminus. 157-165 The / A02 nanobody sequence specifically recognizes two different antigens and targets immune effector cells to tumor cells, thereby increasing the effectiveness of immune effector cells in killing tumor cells.

[0007] This invention provides a bispecific antibody targeting intracellular NY-ESO-1, the bispecific antibody comprising:

[0008] The first antibody that specifically binds to the first antigen

[0009] And a second antibody that specifically binds to the second antigen;

[0010] The first antigen is CD3 and the second antigen is the tumor-specific antigen NY-ESO-1;

[0011] The first antibody is a full-length antibody, consisting of two heavy chains and two light chains.

[0012] The second antibody is a VHH nanobody, and the second antibody is attached to one end of the heavy chain or light chain of the first antibody.

[0013] The tumor-specific antigen NY-ESO-1 is specifically the tumor-specific antigen NY-ESO-1. 157-165 / A02.

[0014] Preferably, the bispecific antibody comprises one or more of the aforementioned VHHs, wherein the VHHs are linked to the N-terminus of the heavy chain of the first antibody via a linker. The amino acid sequence of the linker is GGGGSGGGGSGGGGS.

[0015] More preferably, the bispecific antibody comprises one of the VHHs, the amino acid sequence of which is shown in SEQ ID No. 9.

[0016] The first antibody is either monoclonal antibody OKT3 or monoclonal antibody V9.

[0017] Specifically, when the first antibody is the monoclonal antibody OKT3, the heavy chain amino acid sequence is shown in SEQ ID No. 1, and the light chain amino acid sequence is shown in SEQ ID No. 2;

[0018] When the first antibody is monoclonal antibody V9, the heavy chain amino acid sequence is as shown in SEQ ID No. 3, and the light chain amino acid sequence is as shown in SEQ ID No. 4.

[0019] The present invention also provides a gene encoding the bispecific antibody, the gene sequence encoding VHH being shown in SEQ ID No. 10.

[0020] When the first antibody is the monoclonal antibody OKT3, the gene sequence encoding the heavy chain is shown in SEQ ID No. 5, and the gene sequence encoding the light chain is shown in SEQ ID No. 6.

[0021] When the first antibody is monoclonal antibody V9, the gene sequence encoding the heavy chain is shown in SEQ ID No. 7, and the gene sequence encoding the light chain is shown in SEQ ID No. 8.

[0022] The present invention also provides a method for preparing the bispecific antibody, comprising the following steps:

[0023] (1) Construct a vector that encodes the heavy chain of the first antibody, the light chain, and the VHH encoding gene;

[0024] (2) Transfect the vector expressing the heavy chain of the first antibody, the light chain, and the VHH encoding gene of step (1) into mammalian cells, culture them, and then purify the protein to obtain the bispecific antibody.

[0025] Preferably, the vector in step (1) is pcDNA3.1(+); and the mammalian cell in step (2) is HEK293F cell.

[0026] The present invention also provides the use of the aforementioned bispecific antibody in the preparation of drugs for antitumor or anti-autoimmune diseases.

[0027] The present invention also provides the use of the gene or cells containing the gene in the preparation of drugs for antitumor or anti-autoimmune diseases.

[0028] The beneficial effects of this invention are:

[0029] This invention designs a bispecific antibody targeting intracellular NY-ESO-1, which retains the complete molecular structure of CD3 monoclonal antibodies in a symmetrical form, while adding anti-NY-ESO-1 at the N-terminus. 157-165 The / A02 nanobody sequence can specifically recognize two different antigens. The bispecific antibody provided by this invention is target-dependent, significantly upregulates early and late T cell activation markers, and exhibits significant specific in vivo antitumor activity. Attached Figure Description

[0030] Figure 1This is a schematic diagram of bispecific antibodies; where ab represents two forms of bispecific antibodies, named VHH-CD3 and VHH-CD3V9, respectively.

[0031] Figure 2 The image shows the SDS-PAGE analysis results of the bispecific antibody VHH-CD3; lane 1 is the non-reduced sample, and lane 2 is the reduced sample.

[0032] Figure 3 The image shows the SDS-PAGE analysis results of the bispecific antibody VHH-CD3V9; lane 1 is the non-reduced sample, and lane 2 is the reduced sample.

[0033] Figure 4 Figure 1 shows the results of the bispecific antibody VHH-CD3 lactate dehydrogenase (LDH) detection; Figure 2a shows the results of the A375NY detection in tumor cells, and Figure 3b shows the results of the A375 detection.

[0034] Figure 5 Figure 1 shows the detection results of bispecific antibody VHH-CD3V9 lactate dehydrogenase (LDH); Figure 2a shows the detection results of tumor cells A375 NY, and Figure 3b shows the detection results of A375.

[0035] Figure 6 The figure shows the results of upregulation of T cell activation markers CD69 and CD25 mediated by the bispecific antibody VHH-CD3.

[0036] Figure 7 The figure shows the upregulation of CD69 and CD25, markers of T cell activation, mediated by the bispecific antibody VHH-CD3V9: where ab represents the upregulation of CD69 and CD25, respectively.

[0037] Figure 8 The graph shows the in vivo antitumor activity results of the bispecific antibody VHH-CD3; ** indicates p < 0.01, *** indicates p < 0.001.

[0038] Figure 9 Figure showing the in vivo antitumor activity of the bispecific antibody VHH-CD3V9; * indicates p < 0.05, ** indicates p < 0.01. Detailed Implementation

[0039] Example 1: Expression and Purification of Bispecific Antibody

[0040] The two bispecific antibody gene sequences of this invention were synthesized by Genscript Biotech. The gene sequence encoding VHH is shown in SEQ ID No. 10, the gene sequence encoding one heavy chain is shown in SEQ ID No. 5, the gene sequence encoding the light chain is shown in SEQ ID No. 6, the gene sequence encoding the other heavy chain is shown in SEQ ID No. 7, and the gene sequence encoding the light chain is shown in SEQ ID No. 8. VHH is linked to the N-terminus of the heavy chain of the first antibody via a linker (GGGGS)3. The two bispecific antibody genes were inserted into the EcoRI and NotI restriction sites of the vector pcDNA 3.1(+) to obtain expression plasmids for the two bispecific antibodies. HEK293F cells were transfected with the expression plasmids for 3-4 days, centrifuged at 4000×g for 20 min, and the cell supernatant was filtered through a 0.22 μm filter and purified using an AKTA protein purifier and a HiTrap protein A affinity column. The purification steps are as follows: a. Turn on the AKTA protein purifier and the connected control computer. After the instrument is connected to the computer, set the pressure parameters (high pressure 0.25 MPa); b. Place pump heads A and B in pure water filtered through a 0.45 μm filter membrane, set the flow rate (3 mL / min) and the rinsing ratio of pumps A and B (50% B). After the pure water reaches conductivity equilibrium (approximately 60 mL), connect the HiTrap protein A affinity column (5 mL) to the AKTA purifier and continue rinsing with pure water for at least three column volumes; c. Change the rinsing ratio of pumps A and B to 0% B, and replace pump A with 50 mM Tris-HCl (pH 7.4) loading buffer. After conductivity equilibrium, replace pump A with the supernatant of the culture medium to be purified. After loading, replace pump A with loading buffer until conductivity equilibrium is reached; then change the ratio of pumps A and B to 100% B, and replace pump B with 1 mol / L sodium acetate (pH 7.4). 3.0) The target protein can be obtained by elution in the elution buffer. The AKTA purification instrument is rinsed with pure water until conductivity equilibrium is reached and the entire system is stored in 20% ethanol (v / v).

[0041] Different fusion protein forms were ultrafiltered using 50 kDa ultrafiltration membranes. After replacing the solvent with PBS buffer, protein concentrations were determined using a Nanodrop ND-1000, and the proteins were aliquoted and stored at -80°C for later use. Protein purity was analyzed by SDS-PAGE, and the results are shown below. Figure 2 and Figure 3 As shown.

[0042] Example 2: Bispecific antibody-mediated killing of tumor cells by PBMCs

[0043] Lactate dehydrogenase (LDH) detection.

[0044] A375 is an endogenous expression of NY-ESO-1. 157-165 The A02 melanoma cell line has low antigen expression. A375NY is constructed by stably transfecting A375 cells with EGFP-ubiquitin-SLLMWITQC and constructing a fluorescently labeled NY-ESO-1. 157-165 Tumor cell lines with high A02 expression.

[0045] The disruption of cell membrane structure caused by apoptosis or necrosis leads to the release of enzymes from the cytoplasm into the culture medium, including lactate dehydrogenase (LDH), an enzyme with relatively stable activity. Quantitative analysis of cytotoxicity can be achieved by detecting the activity of LDH released from ruptured cells into the culture medium.

[0046] PBMC was purchased from Shanghai Saili Biotechnology. PBMC and NY-ESO-1 157-165 / A02 pMHC-positive tumor cells A375 and A375NY were mixed at an effector-to-target ratio of 4:1 and seeded into 96-well plates. A gradient concentration of bispecific antibody was added to a final volume of 100 μl (n=3). The cell culture medium was phenol red 1640 medium with 1% FBS to avoid interfering with the final results of the LDH assay. After incubation in a 5% CO2, 37°C cell culture incubator for 24 or 48 hours, the LDH content released after tumor cell death was detected using an LDH detection kit (Tongren Chemical, CK12) to characterize tumor cell mortality. The IC50 was calculated, and the antitumor activity of different forms of bispecific antibodies was compared.

[0047] The bispecific antibody VHH-CD3 exhibited dose-dependent cytotoxic effects against both the naturally NY-ESO-1-expressing A375 cell line and cell lines that overexpress A375. Figure 4 The bispecific antibody VHH-CD3V9 exhibited dose-dependent cytotoxic effects on both the naturally expressing NY-ESO-1 cell line A375 and cell lines overexpressing A375, while the control monoclonal antibody showed weak cytotoxic activity. This indicates that the cytotoxic effect of the bispecific antibody is not non-specific killing caused by T cell activation, but rather target-dependent. Figure 5 ).

[0048] Example 3: Detection of Bispecific Antibody-Mediated T Cell Activation

[0049] Tumor cells A375 and PBMCs were mixed at an effector-to-target ratio of 4:1 and seeded into 48-well plates. A series of gradient concentrations of bispecific antibody diluted in RPMI-1640 complete medium were added to a final volume of 300 μL (n=3), and the plates were incubated for 40 h. Cells were collected by centrifugation at 600×g for 5 min, washed once with 1×PBS, and resuspended in 100 μL of 1×PBS. 5 μL of FITC-labeled mouse anti-human CD3 antibody (BD, 555339), 5 μL of APC-labeled mouse anti-human CD69 antibody (BD, 555533), and 1 μL of PE-labeled mouse anti-human CD25 antibody (BD, 555432) were added, and the plates were incubated at 4°C in the dark for 30 min. After washing twice with 1×PBS, the plates were resuspended in 200 μL of 1×PBS and incubated with ACEA NovoCyte. TM The proportion of CD69 and CD25 positive T cells was detected by flow cytometry.

[0050] The bispecific antibody VHH-CD3 significantly upregulated CD69, an early T cell activation marker, and CD25, a late T cell activation marker. Figure 6 The bispecific antibody VHH-CD3V9 significantly upregulated CD69, an early T cell activation marker, and CD25, a late T cell activation marker, while the control CD3 monoclonal antibody did not induce T cell activation. Figure 7 )

[0051] Example 4: In vivo antitumor activity of bispecific antibody VHH-CD3

[0052] Jicui Pharmaceutical purchased 15 severely immunodeficient NOD-SCID mice aged 5-7 weeks and weighing approximately 20g, and subcutaneously inoculated them with A375 cells (2×10⁶ cells per 10⁻ ... 6 (1 cell / each), until the tumor grows to 100mm 3 Mice were randomly divided into three groups: PBS group, VHH-CD3 group, and CD3 monoclonal antibody (OKT3 monoclonal antibody, heavy chain amino acid sequence as shown in SEQ ID No. 11, light chain amino acid sequence as shown in SEQ ID No. 2) group. Each mouse was intraperitoneally injected with human PBMC (1×10⁻⁶). 7 A transient humanized tumor-bearing mouse model was constructed using immune cells (1 cell / mouse). Subsequently, except for the PBS group, the other two groups of mice were administered 0.5 mg / kg of the control drug and the experimental drug via intraperitoneal injection every 3 days for a total of 3 administrations. Tumor size (length and width, tumor size = length × width × width / 2) and body weight were measured every 3 days. After three administrations, the tumor volume in the VHH-CD3 group showed a significant difference compared to the PBS group (P < 0.001) and also a significant difference compared to the CD3 monoclonal antibody group (P < 0.01). Figure 8This indicates that the bispecific antibody VHH-CD3 has significant specific in vivo antitumor activity.

[0053] Example 5: In vivo antitumor activity of bispecific antibody VHH-CD3V9

[0054] Jicui Pharmaceutical purchased 20 severely immunodeficient NOD-SCID mice aged 5-7 weeks and weighing approximately 20g, and subcutaneously inoculated them with A375 cells (2×10⁶ cells per 10⁻ ... 6 (cells / each), until the tumor grows to 40mm 3 Mice were randomly divided into four groups: PBS group, VHH-CD3V9 0.1 mg / kg group, VHH-CD3V9 1 mg / kg group, and UCHT1 V9 monoclonal antibody (heavy chain amino acid sequence as shown in SEQ ID No. 12, light chain amino acid sequence as shown in SEQ ID No. 4) 1 mg / kg group. Each mouse was intraperitoneally injected with human PBMC (1 × 10⁻⁶ mg / kg). 7 A transient humanized tumor-bearing mouse model was constructed using immune cells (1 cell / mouse). Subsequently, except for the PBS group, the other three groups of mice received intraperitoneal injections of 0.1 mg / kg or 1 mg / kg of the control drug and experimental drug, respectively, every 3 days for a total of 6 administrations. Tumor size (length and width, tumor size = length × width × width / 2) and body weight were measured every 3 days. After six administrations, the tumor volume in the VHH-CD3V9 1 mg / kg group showed a significant difference compared to the PBS group (P < 0.001) and also a significant difference compared to the V9 monoclonal antibody 1 mg / kg group (P < 0.01). Figure 9 This indicates that the bispecific antibody VHH-CD3V9 has significant specific in vivo antitumor activity.

Claims

1. A bispecific antibody targeting intracellular NY-ESO-1, characterized in that, The bispecific antibody comprises: The first antibody that specifically binds to the first antigen. And a second antibody that specifically binds to the second antigen; The first antigen is CD3 and the second antigen is the tumor-specific antigen NY-ESO-1; The first antibody is a full-length antibody, consisting of two heavy chains and two light chains. The second antibody is a VHH nanobody; The bispecific antibody contains two VHHs, which are respectively linked to the N-terminus of the two heavy chains of the first antibody via linkers. The amino acid sequence of the linker is GGGGSGGGGSGGGGS. The amino acid sequence of the VHH is shown in SEQ ID No. 9; The first antibody is either monoclonal antibody OKT3 or monoclonal antibody V9; When the first antibody is the monoclonal antibody OKT3, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID No. 1, and the light chain amino acid sequence is as shown in SEQ ID No. 2; When the first antibody is monoclonal antibody V9, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID No. 3, and the light chain amino acid sequence is shown in SEQ ID No.

4.

2. The gene encoding the bispecific antibody of claim 1, wherein the gene sequence encoding VHH is shown in SEQ ID No.

10. When the first antibody is the monoclonal antibody OKT3, the gene sequence encoding the heavy chain of the bispecific antibody is shown in SEQ ID No. 5, and the gene sequence encoding the light chain is shown in SEQ ID No.

6. When the first antibody is monoclonal antibody V9, the gene sequence encoding the heavy chain of the bispecific antibody is shown in SEQ ID No. 7, and the gene sequence encoding the light chain is shown in SEQ ID No.

8.

3. The method for preparing the bispecific antibody as described in claim 1, characterized in that, Includes the following steps: (1) Construct a vector that encodes the heavy chain of the first antibody, the light chain, and the VHH; (2) Transfect the vector expressing the heavy chain of the first antibody, the light chain, and the VHH encoding gene of step (1) into mammalian cells, culture them, and then purify the protein to obtain the bispecific antibody.

4. The preparation method according to claim 3, characterized in that, The vector in step (1) is pcDNA3.1(+); the mammalian cell in step (2) is HEK293F cell.

5. The application of the bispecific antibody as described in claim 1 in the preparation of an antitumor drug, wherein the tumor is melanoma.

6. The use of the gene or cells containing the gene as described in claim 2 in the preparation of an antitumor drug, wherein the tumor is melanoma.

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